Nema ology, 2004, Vol. 6(6), 911-917
Spon aneous and induced ac i i y o He e o habdi is megidis
in ec i e ju eniles du ing s o age
Paul F.L. FITTERS ∗and Ch is ine T. GRIFFIN
Ins i u e o Bioenginee ing and Ag oecology and Depa men o Biology,
Na ional Uni e si y o I eland, Maynoo h, Co. Kilda e, I eland
Recei ed: 24 Sep embe 2004; e ised: 5 No embe 2004
Accep ed o publica ion: 5 No embe 2004
Summa y – In ec i e ju eniles (IJ) o h ee He e o habdi is megidis isola es, HF85, EU17 and UK211, we e s o ed in wa e a 20◦C o
up o 10 weeks. A 2-week in e als, ac i i y, in ec i i y, ene gy ese es and su i al we e measu ed. The e was no di e ence be ween
he h ee isola es in in ec i i y, which inc eased signi ican ly o e he i s 2 weeks and declined g adually he ea e . IJ became inac i e
du ing s o age. Ou o s o age, he highes ac i i y was eco ded in week 0: nea ly all IJ we e ac i e wi hin he i s minu e o obse a ion
and emained ac i e o he 20-min obse a ion pe iod. Wi h inc easing s o age ime, an inc easing p opo ion o IJ we e inac i e in he
i s minu e, eaching 83-96% by week 6. The ime aken by 50% o he IJ o become ac i e (AT50) ini ially inc eased wi h nema ode
age, eaching a maximum o 3-7 min in week 4 o 6 (depending on isola e) bu subsequen ly declined o 2-4 min in week 10. By he
ime he IJ we e becoming mo e eadily ac i a ed in weeks 6-8, 75% o he lipid ese es had been deple ed, and IJ had s a ed o die.
This g ea e p opensi y o become ac i e wi h age may ep esen a swi ch o isk- aking beha iou in he ace o impending s a a ion.
Keywo ds – ac i i y, en omopa hogenic nema ode, phased in ec i i y, s a a ion, su i al.
The in ec i e ju enile (IJ) o en omopa hogenic nema-
odes (S eine nema and He e o habdi is) is a non- eeding
s age adap ed o hos inding and o su i al ou side he
hos . They ha e conside able ene gy ese es, mainly neu-
al lipid which can cons i u e up o 40% o he IJ weigh
(Sel an e al., 1993; Abu Ha ab & Gaugle , 1997; Fi -
e s e al., 1999). These ex ensi e ese es enable hem
o su i e o weeks o e en mon hs in soil o s o age
(Pa el e al., 1997; Qiu & Bedding, 2000; Hass e al.,
2002).
I has been no ed ha IJ o en omopa hogenic nema-
odes become inac i e in wa e , p esumably o conse e
ene gy (Ishibashi & Kondo, 1990). The p opensi y o be-
come inac i e in wa e a ies be ween species: he pe -
cen age o IJ inac i e in wa e was much g ea e o S ei-
ne nema ca pocapsae han o ei he S. glase i o He e-
o habdi is bac e iopho a (Lewis e al., 1995). Dempsey
and G i in (2002) s o ed H. megidis (UK211) in wa e
a 20◦C o 4 weeks and eco ded he pe cen age o nema-
odes ac i e in samples aken a in e als. The pe cen age
o ac i e IJ declined o e he 4-week pe iod bu a no ime
did i d op below 70% (Dempsey & G i in, 2002). Com-
∗Co esponding au ho , e-mail: [email p o ec ed]
P esen add ess: Teagasc, College o Ameni y Ho icul u e, Bo anic Ga dens, Glasne in, Dublin 9, I eland.
pa able esul s we e ound o S. glase i and H. bac e io-
pho a in simila expe imen s (Lewis e al., 1995). This
aises he ques ion whe he , and o wha ex en , IJ o hese
species become inac i e when s o ed a oom empe a u e
o pe iods anging be ween 1-10 weeks. The objec i e o
bo h he Lewis e al. (1995) and he Dempsey and G i -
in (2002) s udies was o documen age- ela ed changes in
he ac i i y o IJ obse ed unde cons an condi ions, and
no o asce ain wha p opo ion o IJ became inac i e du -
ing s o age in wa e . The la e is impo an because con-
se ing ene gy by becoming inac i e could ex end hei
li e span. To da e he ac i i y s a us o IJ undis u bed du -
ing p olonged s o age has no been in es iga ed o any
species o en omopa hogenic nema ode.
I is well es ablished ha in ec i e ju eniles o many
pa asi ic nema odes become inac i e in he absence o
s imula ion and espond o a change in in ensi y o s imuli
o a ious modali ies ( he mal, mechanical, chemical) by
becoming ac i e (C oll, 1972; C oll & Al-Hadi hi, 1972).
A ypical egime o ac i i y ollows: he pe cen age o
nema odes ac i e and he a e o undula ions quickly
each a maximum, and hen g adually decline. The shape
©Koninklijke B ill NV, Leiden, 2004 911
Also a ailable online - www.b ill.nl
P.F.L. Fi e s & C.T. G i in
o his ac i i y cu e is di e en o di e en nema ode
species and depends on he ype and in ensi y o he
s imulus, he age o he nema ode and he his o y o
s o age (C oll & Al-Hadi hi, 1972). A measu ed esponse
is ecologically impo an o su i al, bo h long e m
(ene gy conse a ion) and sho e m (quick eac ion o
hos s o p eda o s).
In his s udy we desc ibe he spon aneous and induced
ac i i y o s o ed H. megidis IJ. We also assess o he im-
po an pa ame e s o en omopa hogenic nema odes ha
ypically change du ing s o age: su i al, lipid ese es
and in ec i i y (Lewis e al., 1995; Pa el e al., 1997;
Dempsey & G i in, 2002).
Ma e ials and me hods
NEMATODE CULTURES
Th ee no h wes Eu opean H. megidis isola es we e
chosen based on a ied pe sis ence and lipid deple ion
a es in p e ious expe imen s (Fi e s & G i in, unpubl.).
They we e: UK211 (UK), HF85 (The Ne he lands) and
EU17 (Es onia). They we e p opaga ed in la ae o he
g ea e wax mo h, Galle ia mellonella (The Mealwo m
Company, She ield, UK). Ha es ed IJ we e washed
h ee imes by sedimen a ion in ap wa e . Nema odes
(1000 IJ/ml) we e s o ed in 5 cm diam. Pe i dishes
(10 ml/dish) sealed wi h Pa a ilm®. Eigh een dishes o
each isola e we e s o ed a 20◦C in he da k in a an-
domised block design, each block con aining one dish
( eplica e) pe isola e. Ac i i y, su i al and in ec i i y
we e assessed a e 1 day (week 0) and a e 2, 4, 6, 8 and
10 weeks. Th ee blocks we e assessed on each da e. En-
e gy ese es o IJ we e assessed a weeks 0, 4 and 8. The
expe imen was un h ee imes using di e en nema ode
cul u e ba ches o each expe imen al un.
ACTIVITY
The main objec i e o he s udy was o asce ain
whe he IJ ha had been s o ed undis u bed o up o
10 weeks emained spon aneously ac i e. A seconda y
ques ion was whe he he ime aken by inac i e IJ o
become ac i e changed o e ime. Dis u bance was min-
imised by main aining he same empe a u e o s o age
and obse a ion. Mechanical s imula ion o he nema odes
and he ime be ween s o age and obse a ion we e also
minimised. One dish a a ime was ca e ully emo ed om
he incuba o , shaken once o suspend he nema odes, and
17 µl o he suspension was placed in a 16 mm2well o
acili a e simul aneous obse a ion o all IJ in he sam-
ple (a e age 15; ange 10-25). The well was a sub-well
o a 24 well cloning pla e (S e ilin, S one, UK; 16 sub-
wells/well). The mul i-well pla e was closed and placed
on he s age o a dissec ing mic oscope (Nikon SMZ-U,
4×objec i e) illumina ed wi h ib e op ic (‘cold’) ligh
(In alux 4000-1, Volpi, Swi ze land). Room empe a u e
was 20 ±1◦C. The beha iou o he IJ was eco ded o
20 min using a ideo came a moun ed on he mic oscope.
Reco ding began wi hin 30 s o he IJ being aken om
s o age. Ac i i y was assessed in min 1 (0-60 s) 5, 10, 15
and 20. The 1 min eading ga e an es ima e o spon a-
neous ac i i y. In each 1-min assessmen pe iod, each IJ
was sco ed as being inac i e, mode a ely ac i e o highly
ac i e, based on he occu ence o he a ious beha iou s
desc ibed by Dempsey and G i in (2002). Beha iou s
classed as highly ac i e we e: wa ing (backwa ds di ec ed
wa es ha esul in o wa d mo emen ), head h us ing
and head li ing. Beha iou s classed as mode a ely ac i e
we e body mo emen s ( wi ching and non-smoo h mo e-
men s), coiling and e e se wa e. The la e beha iou s
a e ypical o IJ ha a e becoming ac i e, al hough hey
may also be seen in ully ac i e IJ in e spe sed be ween
bou s o wa ing. IJ ha emained inac i e h oughou he
20 min assessmen pe iod we e p obed wi h a dissec ing
needle. Those ha ailed o espond we e classed as dead
and we e no included in he beha iou al analysis.
SURVIVAL,ENERGY RESERVES AND INFECTIVITY
Following assessmen o ac i i y, he IJ suspension o
he h ee eplica e dishes was pooled. Su i al o IJ was
assessed om coun s o h ee o i e eplica e 100 µl
aliquo s.
Ene gy ese es o IJ we e es ima ed by image analysis
densi ome y as desc ibed by Fi e s e al. (1997). Op ical
densi y pe uni a ea (OD uni s/µm2), o mean g ey le el,
is a good co ela e o lipid le els in en omopa hogenic
nema odes (Fi e s e al., 1997; Qiu & Bedding, 1999).
P io o assessmen , IJ we e hea -killed by subme sion
in wa e a 80◦C. Measu emen s we e pe o med on 30
nema odes/ eplica e.
In ec i i y was es ed using a me hod simila o ha
o Dempsey and G i in (2002). One hund ed IJ we e
added o he op o a 55 mm high ial packed wi h mois
sand wi h a G. mellonella la a a he bo om. Vials we e
incuba ed o 24 h a 20◦C. The numbe o IJ ha had
in aded he insec s was assessed by dissec ion 5 days la e .
The e we e en eplica e ials pe ea men .
912 Nema ology
Ac i i y o He e o habdi is megidis in ec i e ju eniles du ing s o age
STATISTICS
S a is ical analyses we e pe o med using Sigma S a
o Windows 1.0 (Jandel 1992-1994). Tes s used we e
ANOVA (one o wo-way) ollowed by an all pai wise
mul iple compa ison p ocedu e (S uden -Newman-Keuls
(SNK), P<0.05).
P obi analysis (SPSS® o Windows™, Release 6.0)
was used o calcula e he 50% ac i a ion ime (AT50:
he ime ou o s o age a e which 50% o he IJ
popula ion was ac i e), 50% su i al ime (ST50), and
he ime by which 50% o a ailable ene gy ese es
we e deple ed (ET50). Ene gy ese es, measu ed as
op ical densi y, anged om he maximum o 0.54 OD
uni s/µm2(week 0) o an asymp o ic minimum o 0.36
OD uni s/µm2. The di e ence be ween hose wo alues,
a e aging 0.18 uni s/µm2, ep esen s a ailable ene gy
ese es (Fi e s & G i in, unpubl.). ET50 and AT50
analysis was pe o med on he log10- ans o med da a,
sepa a ed o each isola e and week (P < 0.05).
Resul s
ACTIVITY
A e 1 day in s o age, 95-100% o H. megidis IJ we e
ac i e du ing he i s minu e hey we e obse ed (Fig. 1,
week 0). In min 1, a high p opo ion o IJ (32-60%)
we e mode a ely ac i e, bu a e 5 min his beha iou
was displayed by less han 16% o he popula ion and
he emainde we e highly ac i e. Wi h inc easing s o age
ime, an inc easing pe cen age o IJ was inac i e in he
i s minu e and a dec easing pe cen age o IJ became
highly ac i e a any ime du ing he 20 min assessmen .
By week 6, ewe han 17% o IJ we e ac i e du ing he
i s minu e and no mo e han 40% we e highly ac i e
a any ime du ing he obse a ion. Fo each o he h ee
isola es, he ime equi ed o 50% o IJ o become ac i e
(AT50) ended o inc ease wi h s o age ime o e he i s
4-6 weeks, bu dec eased wi h u he s o age (Table 1).
Fo bo h HF85 and UK211 he AT50 in week 10 was
signi ican ly lowe han in week 6 (based on non-o e lap
o iducial limi s) showing ha he nema odes became
ac i e as e in week 10 han in week 6.
Th oughou he expe imen , he pe cen age o UK211
ha became ac i e ended o be lowe han o ei he EU17
o HF85 (Fig. 1). A mo e ma ked end was he lowe
pe cen age o UK211 ha was highly ac i e. When s o ed
o 4 weeks o longe , usually 40-50% o he popula ion o
EU17 and HF85 was highly ac i e a e 20 min, while o
Table 1. Fi y pe cen ac i a ion ime (AT50) (95% iducial
limi s) o he in ec i e ju eniles (IJ) o He e o habdi is megidis
isola es HF85, UK211 and EU17. IJ we e s o ed a 20◦C o up
o 10 weeks p io o obse a ion.
S o age ime AT50 (min)
(weeks) EU17 UK211 HF85
00.00
1) 0.001) 0.001)
2 0.77 1.90 0.55
(0.24-1.39) (0.19-3.97) (0.25-0.89)
4 2.04 4.65 4.90
(1.21-2.90) (3.48-5.92) (2.34-8.01)
6 3.15 7.29 3.78
(2.02-4.37) (5.59-9.09) (3.04-4.56)
8 2.47 4.79 2.54
(1.71-3.28) (3.13-6.56) (1.53-3.63)
10 1.90 4.06 1.94
(1.08-2.79) (2.96-5.22) (1.25-2.71)
1) Close o 100% o IJ we e ac i e wi hin he i s minu e.
UK211 his was always less han 20%. The 50% ac i a ion
ime o UK211 ended o be longe han o HF85 o
EU17, and in weeks 6 and 10 he di e ence be ween
UK211 and he o he wo isola es was signi ican based
on he non-o e lap o 95% iducial limi s (Table 1).
SURVIVAL,ENERGY RESERVES AND INFECTIVITY
Mo e han 80% o he IJ o each isola e emained
ali e in week 6 (Fig. 2). A weeks 8 and 10, he e we e
signi ican ly (P < 0.05) ewe HF85 IJ ali e compa ed o
UK211 and EU17, and his is e lec ed in a signi ican ly
sho e ST50 o his isola e (Table 2).
The ime aken o 50% o he ese es o be deple ed
(ET50) anged om 3.2 o 5.4 weeks (Table 2). HF85
had he lowes ET50 e lec ing mo e apid deple ion
o ese es bu di e ences be ween isola es we e no
signi ican as he iducial limi s o e lapped (Table 2). By
week 8, an es ima ed 75% o mo e o he ene gy ese es
o all h ee isola es we e deple ed (da a no shown).
The h ee isola es did no di e in in ec i i y a any
assessmen da e (ANOVA, P>0.05). O e all, in ec i i y
showed a signi ican wo- old inc ease du ing he i s
2 weeks o s o age and declined he ea e (Fig. 3).
Discussion
This is he i s a emp o documen he deg ee o which
he IJ o a species o en omopa hogenic nema ode emain
spon aneously ac i e when s o ed undis u bed in wa e
Vol. 6(6), 2004 913
P.F.L. Fi e s & C.T. G i in
Fig. 1. Pe cen age o highly ac i e, mode a ely ac i e and inac i e li ing in ec i e ju eniles o He e o habdi is megidis isola es HF85,
UK211 and EU17 du ing he i s 20 min a e emo al om s o age a weeks 0, 2, 4, 6, 8 and 10. Each ba ep esen s he mean o h ee
expe imen s.
914 Nema ology
Ac i i y o He e o habdi is megidis in ec i e ju eniles du ing s o age
Fig. 2. Pe cen age o IJ ali e o He e o habdi is megidis iso-
la es HF85, UK211 and EU17 du ing s o age in ap wa e
a 20◦C o 10 weeks. Wi hin weeks, same le e s indica e no sig-
ni ican di e ence be ween isola es (ANOVA, SNK, P<0.05).
Each poin ep esen s he mean o h ee expe imen s (wi h s an-
da d e o o mean).
Table 2. Fi y pe cen su i al ime (ST50) and 50% ene gy
deple ion poin (ET50) (95% iducial limi s) o no h wes
Eu opean He e o habdi is megidis isola es HF85, UK211 and
EU17 s o ed in wa e a 20◦C.
Isola e ST50 (weeks) ET50 (weeks)
HF85 8.26 (7.92-8.64) 3.21 (1.42-4.72)
UK211 10.49 (10.03-11.21) 5.36 (4.36-6.51)
EU17 10.95 (8.69-33.38) 4.45 (3.56-5.37)
Fig. 3. In ec i i y o He e o habdi is megidis isola es HF85,
UK211 and EU17 o Galle ia mellonella in sand. Nema ode
in ec i e ju eniles we e s o ed in ap wa e a 20◦C o up o
10 weeks. Wi hin weeks, same le e s indica e no signi ican
di e ence be ween isola es (ANOVA, SNK, P<0.05). Each
poin ep esen s he mean o h ee expe imen s (wi h s anda d
e o o mean).
o se e al weeks. We show ha H. megidis IJ become
inac i e du ing s o age in wa e , e en a he ela i ely high
s o age empe a u e o 20◦C. I is clea ha om week 4
onwa ds, a mino i y (<40%) o he IJ we e ac i e when
i s obse ed. This may ep esen an o e es ima e o he
le el o spon aneous ac i i y: IJ we e clea ly s imula ed
in o ac i i y, p esumably mainly due o he mechanical
s imula ion o shaking and pipe ing o by ligh exposu e.
A e jus 1 day (week 0), all IJ we e ac i e du ing he
i s minu e o obse a ion. Ei he hey had no become
inac i e a all du ing o e nigh s o age, o hey we e e y
eadily ac i a ed. Wi h inc easing s o age ime, a g ea e
pe cen age o IJ was inac i e du ing he i s minu e
o obse a ion, and hey ook longe o become ac i e.
IJ o en omopa hogenic nema odes gene ally become
less ac i e wi h age, whe he ac i i y is measu ed as
pe cen age ac i e, a e o mo emen o ex en o dispe sal
(Lewis e al., 1995; Wes e man, 1997; Cohen e al.,
2002; Dempsey & G i in, 2002). O he ime-dependen
ac o s such as a p og essi ely inc easing in e al since
las s imula ion, o p og essi e changes in he chemical o
mic obial composi ion o he su ounding medium, may,
in addi ion o ageing, also ha e con ibu ed o he changed
beha iou o he IJ in addi ion o ageing.
I inac i i y is an ene gy-sa ing s a egy aimed a p o-
longing su i al, hen i migh be expec ed ha UK211,
which was he leas eadily ac i a ed o he h ee isola es,
would u ilise i s ese es mo e slowly and su i e longe
han he o he wo isola es. UK211 did lose i s ene gy e-
se es mo e slowly (al hough no signi ican ly) han ei-
he EU17 o HF85, and su i ed signi ican ly longe han
HF85 (al hough no longe han EU17). The con ibu ion
o ac i i y o ene gy expendi u e in nema odes is un e-
sol ed (see W igh , 1998). Howe e , a e o ac i i y o
IJ (measu ed as wa es/min) explained 60% o he a ia-
ion be ween H. megidis isola es in he a e a which lipids
we e deple ed (Fi e s & G i in, unpubl.), implica ing ac-
i i y in ene gy deple ion o hese nema odes.
The inc ease in in ec i i y o each o he h ee H. me-
gidis isola es du ing 2 weeks o s o age a 20◦C con i ms
he phenomenon o he UK211 s ain o H. megidis,as
p e iously epo ed by G i in (1996), Dempsey and G i -
in (2002) and Ryde and G i in (2003). We also ex end
he inding o wo geog aphically dis inc isola es, HF85
om The Ne he lands and EU17 om Es onia. Each o
hese isola es showed he same pa e n o in ec i i y, in-
dica ing ha he e olu iona y p essu es on his ai ha e
been simila o all h ee isola es bu , as all h ee had been
in cul u e o se e al yea s p io o his in es iga ion, in-
Vol. 6(6), 2004 915

P.F.L. Fi e s & C.T. G i in
ad e en labo a o y selec ion may ha e con ibu ed o he
homogenisa ion o his ai . The phenomenon o ‘phased
in ec i i y’ in en omopa hogenic nema odes is con o e -
sial (see e.g., Campbell e al., 1999; Ryde & G i in,
2003) bu he p esen esul s con i m ha he p opo ion
o H. megidis in ading hos s in a ime limi ed-assay un-
de goes a ep oducible inc ease be o e declining.
F eshly ha es ed (week 0) IJ we e nea ly all ac-
i e when i s obse ed. The highly ac i e na u e o
H. megidis immedia ely a e eme ging om he cada e
may e lec he need o dispe se apidly om he highly
c owded en i ons o he cada e . Dempsey and G i in
(2002) showed ha , du ing he i s week a e eme gence,
H. megidis UK211 had high dispe sal a es coupled wi h
low in ec i i y. This dispe si e phase would ensu e ha
no all IJ would in ec local hos s, bu also hos s u he
away, and hus a oid o e c owding. Dempsey and G i in
(2002) desc ibed he ac i i y o H. megidis o e a 4-week
pe iod. They dis inguished h ee phases: an ini ial highly
ac i e phase, an in ec i e phase, and a hi d phase du ing
which in ec i i y and ac i i y we e exp essed a a lowe
le el. As ou obse a ions ex ended o e a longe pe iod
i is possible o dis inguish a u he change in beha iou
o H. megidis. A e 6-8 weeks s o age, he IJ we e mo e
easily ac i a ed han in he p e ious obse a ion pe iod
(weeks 4 o 6, espec i ely) (Table 1). The educ ion in
ac i a ion ime could be a esul o selec i e mo ali y o
less ac i e IJ; howe e , a mos 20% o he IJ had died by
he ime he dec ease in AT50 was eco ded. The al e na-
i e explana ion is ha i ep esen s an inc eased p open-
si y o he gene al popula ion o become ac i e. Possi-
bly, as lipids and o he ese es a e deple ed, IJ swi ch
o a mo e ac i e beha iou , ep esen ing a swi ch o isk-
aking beha iou in he ace o s a a ion (McNama a &
Hous on, 1991). The di e ences be ween isola es in he
ime a which hey became mo e easily ac i a ed is con-
sis en wi h ei he explana ion: i occu ed ea lie (week 6)
o HF85 han o he o he wo isola es, and HF85 also
bo h expe ienced ea lie mo ali y and used up i s ese es
as e .
The ul ima e goal o IJ is o in ec , bu despi e he
inc eased p opensi y o become ac i e a e 6-8 weeks,
hei capaci y o in ec con inued o decline (Fig. 3).
This could be a esul o he dec easing ene gy ese es,
becoming insu icien o eaching and/o en e ing he
hos insec .
Acknowledgemen s
The esea ch was unded by he P og amme o Re-
sea ch in Thi d Le el Ins i u ions (PRTLI) o he Highe
Educa ion Au ho i y, I eland. We a e g a e ul o D
Ca he ine Dempsey o he use ul commen s on he man-
usc ip .
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